The goal of this project is to define the mechanism by which the Ca-sensing receptor (CaR) regulates (inhibits) NaCl transport in the distal nephron, and consequently the mechanism by which it affects blood pressure. Elevated blood pressure is a risk factor for adverse outcomes in cardiovascular, renal diseases and diabetes, all major health problems in the veteran population. In contrast to other G protein-coupled receptors that act via the same G proteins (G1i, G1q, and G113) and that are also located in the distal nephron, the CaR inhibits reabsorption of Na, K, Cl, and water, and so must act via distinct mechasnisms. The overall hypotheses of this application is that the CaR reduces distal nephron ion transport by activating WNK kinases that reduce cell surface expression and activity of Kir4.1, a basolateral K channel, and ClC-Kb, a basolateal Cl channel. This hypothesis is based on preliminary data that demonstrate that: 1) the CaR interacts with Kir4.1; 2) that Kir4.1 interacts with ClC-Kb; 3) that the CaR reduces cell surface expression of Kir4.1; 4) that WNK1 reduces cell surface expression and activity of Kir4.1; 5) that WNK1 siRNA blocks the CaR-dependent reduction in Kir4.1 surface expression and activity; and that 6) all of these proteins are located on the basolateral membrane of the distal nephron. The project has three specific aims: Aim 1. Define the mechanism by which the CaR activates WNK1 and WNK4. Cell surface expression (biotinylation) and channel density (whole cell patch) will be used as measures of CaR signaling through the WNK kinases in HEK-293 cells. Signaling pathways will be analyzed by expressing acitvated and dominant negative protein constructs and siRNAs. Aim 2. Define the effects of the CaR, WNK kinases, and Kir4.1, on the cell surface expression and activity of ClC-Kb (with barttin). These studies will define the interactions of these proteins and will make use of yeast two-hybrid assays and HEK-293 cells for the basic biochemical and electrophysiologic analysis, and Xenopus oocytes for ion-specific measurement of the ClC-Kb and Kir4.1 channel activity measurements. Aim 3. Define the importance of the CaR, WNK kinases, Kir4.1, and ClC-Kb in the control of transepithelial ion transport in polarized renal epithelial cells. The apical membranes of MDCK cell monolayers will be permeabilized, so that the transport characteristics of the the basolateral membrane are measured and the effects of the CaR and WNK kinases on Kir4.1 and ClC-Kb activities can be determined. These studies will define a novel mechansism by which the CaR regulates distal nephron ion transport, will lead to improved understanding of distal nephroin ion transport and blood pressure control, and potentilly improved therapy. PUBLIC HEALTH RELEVANCE: High blood pressure is a risk factor for heart disease, vascular disease, stroke, progression of kidney disease, and many complications of diabetes, all major health probelems for the veteran population. So far, all forms of high and low blood pressure that run in families are caused by processes that affect the amount of salt the kidneys release into the urine or retain in the body. For that reason, understanding how the kidneys retain or release salt is important for understanding control of blood pressure. The goal of this project is to understand how one protein, the calcium receptor (CaR) that senses calcium in the blood and that is present in the kidneys in a region that is important for determining how much salt is retained or released, affects how the kidneys retain or release salt. Some genetic conditions that turn on the CaR and diseases where blood calcium levels are high and activate the CaR to cause loss of salt through the kidneys often resulting in low blood pressure. We found that the CaR uses a group of enzymes called WNK kinases to control two proteins that affect how kidneys retain or release salt. The specific purpose of this application is to determine exacly how the CaR activates the WNK kinases and then how they affect the ability of the kidney to retain or release salt. The first group of experiments will identify the signaling proteins the CaR uses to activate the WNK kinases. These experiments will use simple cultured cells into which the proteins being studied will be introduced. Mutant forms of these proteins that either block or increase their function will be used to determine if they are important. The second set of experiments will test the effects of the CaR and WNK kinases on two proteins that transport salt and will determine if the CaR and WNK kinases affect the amount of salt they can transport. The third set of experiments will make use cultured kidney cells that are grown in a way that allows them to retain or release salt in a manner that is similar to what happens in a kidney. The CaR, the two transport proteins, the WNK kinases, and forms of these proteins that block effects and stimulate their effects will be put into the kidney cells to determine if what was found in the previous experiments applies to kidney cells and the way they normally retain or release salt. This information will result in recognition of a new way to control the amount of salt in the body and hopefully new ways to treat high and low blood pressure.